Spectroscopic probes will be used to test several essential tenets of Mitchell's chemiosmotic coupling theory. The experiments are designed to test if proton movements are associated with the primary steps in energy conservation. Flourochromes which are pH-sensitive will be covalently attached to cytochrome c and "loaded" on the inside of submitochondrial particles to monitor internal pH as a function of membrane energy state. The proton permeability of these membranes will be directly determined by following internal pH responses to external pH changes in the presence and absence of uncouplers. The intenal location of these probes will be tested by the effect of specific antibodies to the purified derivatives on the cytochrome oxidase and reductase activities. Other methods for ascertaining the internal location will focus on the accessibility to membrane impermeable substrates (ferricyanide, cytochrome c peroxidase). The chemical and physical properties of the purified cytochrome derivatives will be characterized, and the site of the probe attachment in the primary sequence will be determined by peptide mapping and amino acid analysis.